The H+-transporting F1Fo ATP synthases of oxidative phosphorylation in mitochondria and bacteria are very similar. Rotation of subunit 3 within the core of the ?3?3 hexamer of F1 drives ATP synthesis by a mechanically driven change in binding affinities for substrates and products. H+ transport through trans- membrane Fo drives rotation of an oligomeric ring of c subunits connected to 3, and results in ATP synthesis in catalytic sites at the interface of 12 subunits. A stator complex of Fo subunits a and b extends from the membrane to the top of the F1 molecule and holds 1323 fixed, relative to the membrane, allowing the c-3 complex to rotate within. The mechanism of coupling H+ transport and c-ring rotation is poorly understood. A high resolution, X-ray structure of a bacterial c-ring has been published and is used here as a model for our structure-function studies with the Escherichia coli enzyme. This proposal focuses on the structure of subunit a and its functional interaction with the c-ring and has the ultimate goal of defining how structural changes at the a-c interface mechanically couple H+ transport with c-ring rotation. The proposed research aims to structurally define trans-membrane helix (TMH) interactions at the subunit a-c interface and the global fold of subunit a in native Escherichia coli membranes, initially by chemical cross-linking and then by more refined dynamic analysis with fluorescence probes. Aqueous access pathways at the subunit a-c interface will be defined via a variety of chemical modifications of cysteine substituted into the TMHs of subunit a and c and by site directed mutagenesis. We hypothesize that these pathways mediate H+ transport from the membrane surfaces to the subunit c aspartate-61 H+ binding site in the middle of the membrane. The mechanism of gating alternate H+ access from the half channels leading to the two sides of the membrane will be probed. We hypothesize that conformational changes in the cytoplasmic loops of subunit a, occurring as a consequence of c-ring rotation, are structurally linked to the swiveling of TMHs in subunit a, and that the swiveling alternatively opens and closes the half channels from aspartate-61 to the two sides of the membrane. The TMH swiveling in subunit a is in turn proposed to mechanically drive c-ring rotation. The ATP synthase is central to cellular function - it makes the ATP. Abnormalities in the enzyme lead to human disease. An understanding of the structure-function interactions at the subunit a-c interface promises to provide insight into the design of new antimicrobial agents that inhibit pathogenic bacteria, e.g. tuberculosis pathogens, without inhibiting the related human mitochondrial F1Fo counterparts. Closely related enzymes are responsible for vesicular acidification in human cells, and work by a similar rotary mechanism. The principles by which this enzyme works may provide fundamental insights into other transport and energy transducing machines that are central in biology and medicine. PUBLIC HEALTH RELEVANCE: ATP is the biological fuel used by all cells to drive all cellular functions. The generation of ATP in cells is catalyzed by a biological rotary motor that is driven by a trans-membrane proton gradient, a motor that can be likened to a water wheel being driven by a stream of water flowing through a stationary source, e.g. a grain mill wheel or a hydroelectric dam. Our research focuses on the interactions of the "water stream and the wheel" and should provide insight into human mitochondrial diseases in which these interactions are defective, and to new classes of drugs/antibiotics that block rotation of the turbine in bacterial cells, but not humans, to inhibit bacterial growth and function.